1. Field of the Invention
The present invention relates to improved artificial diets or growth media for rearing arthropods, including zoophagous arthropods and phytophagous arthropods including facultatively zoophagous phytophages. The growth media of the invention are suitable for mass production of these insects at a reasonable cost for uses including as biological control agents.
2. Description of the Art
The phylum Arthropoda includes insects and arachnids. Within this phylum are zoophagous arthropods (those that eat animal materials), phytophagous arthropods (plant-eating arthropods), and facultatively entomophagous phytophages (plant-eating arthropods that display some animal material consumption in addition to eating plants).
In the United States and throughout the world, the application of synthetic chemical insecticides is the primary method of controlling arthropod pests of many agricultural commodities, including food, fiber, and ornamental crops. However, there is an increasing interest in reducing the use of chemical pesticides and fertilizers and to make agriculture more sustainable. Biological control is recognized as the best alternative to the use of chemical insecticides for controlling these pests.
Use of beneficial arthropod predators and parasites for biological control on a large scale as an alternative to pesticides depends on the ability to mass produce large quantities of viable and biologically fit arthropods at a reasonable cost. However, rearing of beneficial arthropods on their natural hosts/prey or on unnatural factitious hosts is too expensive to allow large scale use of beneficial arthropods in commercial agriculture. Accordingly, artificial diets or growth media are required for mass production at reasonable cost.
The phylum also includes destructive arthropods. For example, the western tarnished plant bug, Lygus hesperus Knight (Hemiptera: Miridae) and tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) are very destructive pests, and their economic impact spans several cropping systems in North America (Hedlund and Graham, USDA Technical Bulletin ARS-64, 1987). Their impact is amplified by their remarkable ability to become resistant to pesticides and by their extremely broad host range (Hedlund and Graham, supra). Therefore, potential alternatives to conventional pesticides to control these pests have become very important. Such alternatives include development of biological control, biorational chemicals, plant breeding, sterile insect release, and genetic engineering. Development of management strategies based on these approaches would depend upon rearing systems that permit medium to large scale rearing of arthropod pests. A major component necessary for such rearing includes an inexpensive, high quality artificial diet that can be used to rear thousands to millions of the targeted pest (Cohen et al. 1999, Biocontrol News and Information, (accepted June, 1999); Nordlund, Biocontrol News and Information 17(2):35-44,1999; Nordlund and Greenberg, Biocontrol News and Information 4:45-50,1994).
A liquid diet for rearing Lygus hesperus which includes whole raw eggs was described by J. W. Debolt, Annals of the Entomological Society of America 75:119-122 (1982). The diet has been used successfully in rearing L. hesperus that were used for rearing both egg and nymphal parasitoids (Debolt, U.S. Dept. Agric. Res. Serv. (ARS-64), pages 82-87, 1987; Debolt, Entomol. Exp. Appl. 50:87-95, 1989). S. A. Hassan and K. S. Hagen, Zeitschrift fur Angewandte Entomologie, 86:315-320 (1978) describe liquid diets for rearing lacewings, Chrysopa rufilabris Stephens, larvae. Semi-solid meat-based artificial diets have been described for rearing Geocoris punctipes (Say) in publications by A. C. Cohen, Journal of Economic Entomology, 78:1173-1175 (1985); A. C. Cohen and N. M. Urias, The Southwestern Entomologist, 11:171-176 (1986); and A. C. Cohen and R. T. Staten in Applications of Genetics to Arthropods of Biological Control Significance, Eds. S. K. Narang et al., CRC Press, Inc., Chapter 7, pp. 121-132 (1994)). De Clercq et al. (Entomophaga 37:149-157 (1992)) describe an artificial insect diet for rearing the predatory stinkbugs Podisus maculiventris and Podisus sagitta using the meat-based diet of Cohen (1985) with added fresh (raw, liquid) egg yolk. Saavedra et al. (MedFac Landbouww Univ Gent 61(3a):767-772 (1996) describe an artificial insect diet for Podisus nigrispinus based on the bovine meat diet developed by Cohen (1985, supra) having added bee""s honey, brewer""s yeast, fresh egg yolk, and Wesson""s salt. An artificial meat paste-based diet containing cooked whole egg, which has been found suitable for mass rearing entomophages (predatory arthropods and parasitic insects) including big-eyed bugs, Geocoris punctipes, and lacewings, Chrysoperla rufilabris, has been described (A. C. Cohen, U.S. Pat. No. 5,834,177). Use of this meat paste-cooked egg diet was also found useful as a supplement for artificial diets for phytophagous pests that are known to supplement their plant-eating habits with some insect consumption (A. C. Cohen, U.S. Pat. No. 5,945,271).
The present invention is directed to improved artificial diets or growth media which are suitable for rearing large numbers of viable and biologically fit arthropods, including zoophagous arthropods and phytophagous arthropods, including facultatively zoophagous arthropods. The diets of the invention are free of insect components and meat products such as meat paste. Because the diets of the invention lack both insect components and meat products (such as meat paste), it was unexpected that they could be used so successfully to produce predatory arthropods and facultatively zoophagous phytophages. Prior art suggests that predatory and parasitic insects and mites require real arthropods or parts from arthropods or at least vertebrate-derived meat products in their immature stages of development.
In a first embodiment, the artificial growth medium is composed of a mixture of cooked egg, liquid, and carbohydrate source. Optional ingredients may be included as described in detail below and as illustrated in the examples. Preferably, the growth medium mixture is sufficiently blended so that the nutrients are substantially compositionally uniformly distributed. This diet is particularly suitable for providing nutrients in amounts and proportions effective to support growth of zoophagous arthropods.
In a second embodiment, the artificial growth medium is composed of a plant-based diet which includes cooked egg yolk or cooked whole egg. Optional ingredients may be included as described in detail below and as illustrated in the examples. This diet is particularly suitable for providing nutrients in amounts and proportions effective to support growth of phytophagous arthropods, including facultatively zoophagous phytophages.
In a third embodiment, the growth medium of the first embodiment is used in combination with a plant-based diet which includes cooked egg yolk or cooked whole egg (growth medium of the second embodiment). Optional ingredients may be included as described in detail below and as illustrated in the examples. This diet is particularly suitable for providing nutrients in amounts and proportions effective to support growth of phytophagous arthropods, including facultatively zoophagous phytophages.
Because the diets of the three embodiments are devoid of both insect components and meat products (such as meat paste), they are less expensive to produce than previously developed comparable diets; simpler to prepare, and lend themselves more readily to prevention of microbial contamination than do previous diets, and thus find particular usefulness for the mass rearing of beneficial and destructive arthropods at a reasonable cost for subsequent uses, including as biological control agents.
Tests of the new diet of the first embodiment with larval green lacewings (Chrysoperla rufilabris) against a control diet described in U.S. Pat. No. 5,834,177 showed that the new diet produces similar quantities of adults of similar longevity rates, with slightly lower weights and numbers of eggs, at a significantly lower cost per kg of diet and with approximately half the labor time. The new diet (approx. $0.75/kg) and the previous medium (approx. $2.00/kg) have been shown to produce green lacewings of similar quality and quantity to insect (Ephestia) eggs, which cost about $300-500/kg.
As discussed in detail, below, the new diet of the third embodiment has been used to rear over fifteen generations of L. hesperus and is currently being used to support production colonies; it has also been used to rear L. lineolaris for five generations, thus far. This indicates the usefulness of the diet for producing high quality, vigorous phytophagous arthropods, including facultatively zoophagous arthropods, while reducing production costs.
The new diet of the third embodiment was compared to the Debolt (1982, supra) diet for rearing the facultatively zoophagous phytophage Lygus hesperus. Biological fitness estimates for L. hesperus indicated that mean biomass production per cage, adult wet and dry weights, survival to the adult stage, and egg production were significantly greater for the new diet than for the existing standard, Debolt (1982) diet. The ingredients in the diet of the third embodiment of the invention cost about xe2x85x9 those in the complex (about 50 ingredients) Debolt diet, and preparation required less than ⅔ of the labor. The cost of diet for production per 1000 eggs was about $0.004 compared to $0.04 for an equal number of eggs from Debolt diet. The new diet further represents an improvement over the Debolt diet because it does not require expensive chemically defined items such as casein, linoleic acid, RNA, defined salts, or pure cholesterol. Instead, it comprises more nutritious components which are presented in a form much more suitable for the extra-oral digestion practiced by species of arthropods such as Lygus and all other Miridae, and nearly all Heteropteraxe2x80x94a slurry of solid materials, including cooked egg, that is blended with the plant components which include cooked egg.
Tests of the new diet of the third embodiment with L. hesperus against a control diet consisting of the phytophagous diet described in of U.S. Pat. No. 5,945,271, showed that the diet of the third embodiment was superior in terms of rates of development, dry weight, egg production, dry weight, and other fitness characteristics.
The diet of the third embodiment of the invention also supports development and reproduction in the tarnished plant bug, L. lineolaris. This is the first time the facultatively zoophagous phytophage Lygus lineolaris was produced for multiple generations using an artificial diet. This was entirely unexpected. Prior to this diet, this species had never been reared beyond a single generation on an artificial diet. It is an extremely difficult insect to colonize, and although it is of great economic and ecological importance as a destructive pest, it has never been reared, even on natural diets, in any substantial enough numbers to be used for biological control programs or other biologically based technologies. Efforts to rear this insect for the past 40 years using an artificial diet were unsuccessful. However, using the diet of the third embodiment of the invention, a colony of L. lineolaris is now being produced at a rate of about 50,000 adults/week, and there appears to be no biological limitation to the potential production of this species on this diet. Thus, the invention represents a major breakthrough in artificial diets for arthropods.
Millions of insects are required for augmentative biological control and other biologically based technologies. Obtaining such quantities demands the use of inexpensive diets to make moderate to large scale operations feasible. The growth media of the invention fulfills this need of providing an economical means for rearing arthropods. The invention also fulfills the need for large scale production of arthropods necessary for technologies such as sterile insect release, production of pathogens and parasitic insects using mass-produced arthropods as food.
Accordingly, the artificial growth media of the invention represent a major breakthrough for large-scale production of arthropods. Because of the high quality of arthropods produced, the lower price of the diets, and the greater ease of microbial decontamination, these diets have the potential to greatly expand the uses of arthropods in agriculture.
The media components are readily available. Further, the media of the invention can be readily packaged, for example in a membrane such as Parafilm(copyright), for presentation to zoophagous arthropods, phytophagous arthropods or facultatively zoophagous phytophages. Additionally, the growth media can be readily freeze-dried, and can be used in the freeze-dried form or used after reconstitution with liquid. The use of freeze-drying (lyophilization) makes the diets easy to store, ship, or handle.
In accordance with this discovery, it is an object of the invention to provide improved artificial diets for production of arthropods, including zoophagous arthropods and phytophagous arthropods, including facultatively zoophagous phytophages.
Another object of the invention is to provide growth media for use for economical mass production of arthropods for subsequent release as biological control agents, for example, augmentative releases for control of populations of insect pests in commercial agriculture.
A further object of the invention is to provide artificial diets for economical large scale production of phytophagous and facultatively zoophagous phytophages for technologies such as sterile insect release, production of pathogens and parasitic insects using mass produced phytophages as food or for use in the production of phytophage products such as entomopathic fungi or entomopathic viruses.
A still further object of the invention is the provision of artificial diets for rearing arthropods which yield high quality predators.
An even still further object of the invention is the provision of artificial diets for rearing insects and other arthropods which are free of insect components and free of meat products.
Other objects and advantages of the invention will become readily apparent from the ensuing description.
The present invention is directed to improved artificial diets or growth media suitable for rearing large numbers of viable and biologically fit zoophagous arthropods and phytophagous arthropods, including facultatively zoophagous phytophages.
In a first embodiment, the artificial growth medium is composed of a mixture of cooked egg, liquid, and carbohydrate source. Optional ingredients may be included as described in detail below and as illustrated in the examples. Preferably, the growth medium mixture is sufficiently blended so that the nutrients are substantially compositionally uniformly distributed. This diet is particularly suitable for providing nutrients in amounts and proportions effective to support growth of eggs, larvae, nymphs of zoophagous arthropods, and also provides nutrients effective for rearing zoophagous arthropods which have predaceous adult stages.
In a second embodiment, the artificial growth medium is composed a plant-based diet which includes cooked egg yolk or cooked whole egg. Optional ingredients may be included as described in detail below and as illustrated in the examples. This diet is particularly suitable for providing nutrients in amounts and proportions effective to support growth of phytophagous arthropods, including facultatively zoophagous phytophages.
In a third embodiment, the growth medium of the first embodiment is used in combination with a plant-based diet which includes cooked egg yolk or cooked whole egg (diet of the second embodiment). Optional ingredients may be included as described in detail below and as illustrated in the examples. This diet is particularly suitable for providing nutrients in amounts and proportions effective to support growth of phytophagous arthropods, including facultatively zoophagous phytophages.
The diets are essentially free of insect components, such as hemolymph, and are xe2x80x9cmeat-free,xe2x80x9d that is, they are essentially free of the edible flesh of animals, for example, the xe2x80x9cmeat pastexe2x80x9d ingredient described in Cohen, 1985, supra, or the xe2x80x9cprotein-lipid pastexe2x80x9d ingredient as described in U.S. Pat. Nos. 5,384,177 or 5,945,271, which patents are incorporated herein in their entirety. Illustrative of the xe2x80x9cmeat pastexe2x80x9d ingredients described in U.S. Pat. Nos. 5,384,177 or 5,945,271 are blended ground beef and beef liver; meat and liver from other animals, such as chicken, lamb, and pork; fish innards; oysters; and cells derived from animals and propagated and multiplied using cloning technology. Because the diets of the invention are free of both insect components and meat paste, it was unexpected that it could be used so successfully to produce predatory arthropods, including predatory insects and facultatively zoophagous phytophages.
Further, because the diets of the invention are devoid of insect components and meat paste, they are useful for the production of arthropods on a commercial scale at a relatively low cost. A further advantage is that the diets lend themselves more readily to prevention of microbial contamination than do previous diets. Moreover, the arthropods are of sufficient quality to function effectively as biological control agents as described above.
As used herein, the term xe2x80x9czoophagous arthropodsxe2x80x9d refers to animals of the phylum Arthropoda that consume animal materials, for example, insects that feed on other insects. Exemplary of zoophagous arthropods are predatory and parasitic insects such as entomophages, and arachnids such as spiders, mites or ticks.
As used herein, the term xe2x80x9centomophagesxe2x80x9d refers to predatory arthropods and parasitic insects (parasitoids). Entomophages are discussed in detail in the text Entomophagous Insects by C. P. Clausen, Hafner Publishing Company, New York (1972), which is incorporated herein by reference. Entomophage refers to insects that feed upon other insects. These insects are broadly divided into two general classes, predators and parasitic insects. While there are many instances of species that are intermediate between the two general classes, a parasitic insect, in general, refers to one that, in its larval stage, develops either internally or externally upon a single host individual, the latter eventually dying as a result. The adults are generally free-living, and their food sources are usually distinct from those of the larvae. In contrast, a predator is generally free-living in the larval stage also and requires a number of individuals to provide food to grow to maturity. Clausen, supra, reports that there are 224 families in 15 orders, which to some extent, feed upon other insects.
Entomophages of particular importance to commercial agriculture are those useful as biological control agents, for example, through augmentative releases, to control populations of insect pests on agricultural commodities. Without being limited thereto, exemplary of predatory arthropods having importance for biocontrol in a commercial agricultural setting include predators of the Order (species)/family: Heteroptera: Geocoris punctipes (Say)/Lygaeidae [big eyed bug]; Podisus maculiventris (Say)/Pentatomidae; Podisus sagitta (Fab.)/Pentatomidae; Macrolophus caliginosus Wagner/Miridae; Neuroptera: Chrysoperla carnea Stephens/Chrysopidae [lacewings]; Chrysopa sinica/Chrysopidae; Chrysopa scelestes Banks/Chrysopidae; Chrysopa lanata lanata Banks/Chrysopidae; Chrysopa septempunctata Wesmael/Chrysopidae; Coleoptera: Coleomegila maculata Harmonia axyridis/Coccinellidae, Olla abdominalis/Coccinellidae. Without being limited thereto, exemplary parasitoids having potential importance for biocontrol in a commercial agricultural setting include Hymenoptera, in particular, Trichogrammatidae; Braconidae, and Ichneumonidae; and Diptera, in particular, Tachinidae.
Exemplary zoophagous arachnids include mites (family Phytoseiidae), and spiders (families Lycosidae, Thomisidae, Linyphiidae, Aranida).
As used herein, the term xe2x80x9cphytophagous arthropodsxe2x80x9d refers to animals of the phylum Arthropoda that consume plants. Without being limited thereto, exemplary of phytophagous arthropods are Lepidoptera such as Helicoverpa zea (cotton bollworm), Heliothis virescens (tobacco budworm), Spodoptera exigua (beet armyworm) and other Spodoptera spp., e.g., Trichoplusia ni (cabbage looper), Pseudoplusia includens (soybean looper); Coleoptera such as Diabrotica spp., Popillae japonica, Epilachna spp.; various Curculionidae (weevils); Hemiptera/Heteroptera such as Lygus spp. (various Miridae), Nezara viridula (southern green stink bug), Euschistus spp.; phytophagous mites (Tetranychidae); Diptera such as Delia platura. 
As used herein, the term xe2x80x9cfacultatively zoophagous phytophagesxe2x80x9d refers to phytophagous (plant-eating) arthropods that display some animal material consumption in addition to eating plants. [See, A. C. Cohen, xe2x80x9cPlant feeding by Predatory Heteroptera: Evolutionary and Adaptational Aspects of Trophic Switching,xe2x80x9d pp. 1-17, 1996, In: Zoophagous Heteroptera: Implications for Life History and Integrated PestManagement, O. Alomar and R. N. Wiedenmann, eds. Thomas Say Publications in Entomology, 1996, and A. C. Cohen, xe2x80x9cBiochemical and Morphological Dynamics and Predatory Feeding Habits in Terrestrial Heteroptera,xe2x80x9d pp. 21-32, In: Predatory Heteroptera in Agroecosystems: Their Ecology and Use in Biological Control, M. Coll and J. R. Ruberson, eds., Thomas Say Publications in Entomology. Entomological Society of America, Lanham, Md., 1998.]
Without being limited hereto, exemplary of predominantly phytophagous insects that display some insect consumption in addition to eating plants, are Heteroptera: Miridae: Lygus spp., for example, Lygus hesperus Knight and L. lineolaris. Lygus spp. are known to be important pests in several cropping systems (seed alfalfa, cotton, strawberries, orchard crops). They are also known to supplement their plant-eating habits with some insect consumption. This fact (their facultative entomophagy) sets the stage for using the growth medium of the second and third embodiments for artificial diet-based production of parasites of phytophagous pests, for example, Lygus bugs. Mass rearing of phytophages is useful for other biocontrol purposes, for example to produce phytophage products such as entomopathic fungi or entomopathic viruses.
Diet of the First Embodiment
The artificial diet or growth medium of the first embodiment of the invention is composed of a mixture of cooked egg, liquid, and carbohydrate source. It is essentially free of insect components and meat paste, as discussed in detail, above. Optional ingredients as described below may be included.
Preparation of the diet is conveniently carried out by blending eggs during the cooking process with carbohydrate solution (liquid plus carbohydrate source) and one or more optional ingredients as desired. The blended egg ingredient in the mixture is cooked from a range of soft gel to hard texture. It is preferred that the egg is cooked to a soft, sticky solid, i.e., non-runny. It appears that this preferred scrambled egg consistency is more advantageous for arthropod production than hard-boiled egg consistency.
Preferably, the growth medium mixture is sufficiently blended so that the nutrients are substantially compositionally uniformly distributed.
The cooked egg ingredient can be provided by whole egg, by yolk and whole egg, by yolk and white, or by yolk alone. The eggs can be, for example, bird eggs such as chicken, turkey, duck, goose, quail, ostrich, or pheasant eggs.
Blended whole egg or blended egg yolk is advantageous for several reasons, including the following: (a) whole egg that is blended and cooked provides a concentrated amount of important nutrients such as cholesterol that do not separate during further handling of the medium, and the heating denatures avidin which otherwise binds biotin, an essential water soluble vitamin, (b) egg yolk provides an excellent natural source of cholesterol, lipids, lipoproteins, protein, and B-vitamins, (c) egg white provides proteins including albumin, (d) egg has hydrophilic and hydrophobic properties, and can retain nutrients by lipid-lipid interactions, protein-protein interactions, hydrophilic interactions, and by the formation of lipoprotein complexes, and (e) it may provide proteoglycans to act as natural cellular adherent materials. Additional egg yoke may also be added to the medium to increase lipoprotein availability.
The liquid component of the medium can comprise water, or other sources of water such as milk, medium from tissue culture or aqueous plant extracts, and may include additional water soluble nutrients or additives such as vitamins, minerals, antimicrobial agents, or preservatives. The liquid functions to support growth of an arthropod, and further, in the case of entomophages that feed using extra-oral digestion, provides liquid for entomophages to pre-digest their food.
The carbohydrate source of the medium can comprise sucrose, plant starch such as corn, potato, wheat, rice, or barley starch, high fructose corn syrup, honey, other plant sugars or animal glycogen or other sugars such as glucose or other monosaccharides; lactose, trehalose or other disaccharides; oligosaccharides or polysaccharides as known to those of skill in the art or combinations thereof. Optionally, glycerol and/or proline or equivalents thereof can be added to the carbohydrate solution as microbial inhibitors.
Other optional adjuvants or supplements may also be incorporated into the medium to enhance the growth of the target arthropod or prevent the growth of microbial contaminants. For example, the growth medium may also include other nutrients, for example, lipids (with or without emulsifying agents) such as plant oils, e.g., corn oil, safflower oil, soybean oil or animal fats such as lard, purified amino acids, and nucleic acids. These nutrients may also have important metabolic and behavior-inducing characteristics, such as phagostimulatory nutrients, that is, an ingredient that stimulates the target arthropod to stimulate the complete feeding response. Exemplary of phagostimulant sources are sucrose, honey, tryptophan, and gamma amino butyric acid.
Optionally, a multivitamin mixture, e.g., Vanderzant vitamin mixture (see E. S. Vanderzant, xe2x80x9cRearing Lygus Bugs on Artificial Diets,xe2x80x9d Journal of Economic Entomology 60:813-816, 1967), other sources of vitamins, or sources of B-vitamins, e.g., brewer""s yeast, torula yeast, may be added to supplement vitamins in the growth medium. Yeast is also a source of nitrogen, amino acids, and trace elements.
Further optional ingredients include preservatives and/or antimicrobial agents. Without being limited thereto, exemplary of such agents are formalin, propionate, potassium sorbate, streptomycin, and chlortetracycline. The antimicrobial agents are included, individually or in combination, in an amount sufficient to prevent growth of microbial contaminants, but insufficient to prevent growth of the target arthropod.
An exemplary formulation is described in detail in Example 1, below. In brief, cooked eggs are blended during the cooking process with sugar solution and honey and stirred with acetic acid and brewer""s yeast. Acetic acid is useful as a food acid for adjusting the pH, and also in helping solidify the egg and help it remain sticky and adherent.
The growth medium provides nutrients in amounts and proportions effective to support growth of zoophagous arthropods, and as discussed in detail, below, in the third embodiment, when used in combination with a plant based diet which includes cooked egg yolk or cooked whole egg, it provides nutrients in amounts and proportions effective to support growth of phytophagous arthropods including facultatively zoophagous phytophages.
In each of the first, second, and third embodiments, the growth medium should contain essential nutrients. Essential nutrients are defined as those nutrients such as minerals, amino acids, cholesterol, fatty acids, lipid soluble vitamins, and water soluble vitamins that are essential to the growth of the target arthropod. This can be readily determined for any circumstance. For instance, the basic nutritional requirements of parasitoids and predators for an artificial growth medium are discussed in S. Grenier et al. in Pest Management in the Subtropics, Biological Controlxe2x80x94a Florida Perspective, Eds. D., Rosen, F. D. Bennett, and J. L. Capinera, Intercept Press, Andover, U.K., Chapter 10, pp. 181-205 (1994), which is incorporated herein by reference. As known to those in the art, nutrients essential for growth of an arthropod can vary among species. For any particular target arthropod, nutrients essential for growth can readily be determined by procedures known to those of skill in the art, for example, dietary deletion. The actual concentrations selected may be determined by the practitioner skilled in the art.
An advantageous feature of diets of the first, second, and third embodiments is that although supplements of vitamins, and preservatives may be added, the diets may be prepared without supplements of defined chemicals such as purified proteins; RNA; purified lipids, e.g., triglyceride, trilinoleic acid; purified salts; or purified amino acids.
In general, when the diet of the first embodiment is used for the production of zoophagous arthropods, the amount of the protein in the growth medium, that is, the combined amount of all protein or combination of amino acids and protein in the medium should be approximately 5-20% of growth medium (total wet weight), the amount of the fat should be about 4-15% of the growth medium (total wet weight), the amount of carbohydrate should be about 3-20% of the growth medium (total wet weight), the amount of cholesterol should be in the range of approximately 250-3000 mg per kg growth medium and the amount of liquid in the growth medium (including added liquid and liquid contained in the other ingredients) should be in the range of about 55-80% of the growth medium (total wet weight). The fat and protein sources may be provided in combination such as by lipoproteins.
In general, when the diet of the first embodiment is used for the production of zoophagous arthropods, the pH of the growth medium can range from about 4.0 to 8.0. It is recommended that the pH of the growth medium for use in rearing entomophages be in the range of approximately pH 5.5 to 7.4. The pH can be conveniently adjusted using a solution of acetic acid or potassium hydroxide.
The medium of the first, second or third embodiment can be readily packaged, for example, in a membrane such as Parafilm(copyright) (a flexible, moldable, self-sealing, odorless, moisture resistant, thermoplastic, semi-transparent, and practically colorless membrane), and be presented to the insects in a shape and wall thickness that simulates natural prey or natural plant materials. The packaged medium can be sterilized or pasteurized to have a longer shelf life for subsequent use for rearing the target arthropod. The growth medium can be readily freeze-dried can be used in the freeze-dried form or used after reconstitution with liquid. The use of freeze-drying (lyophilization) makes the diet easy to store, ship, or handle.
Techniques for rearing arthropods in vitro on artificial diets have been described by Cohen et al., 1985, supra, which is incorporated herein by reference and in U.S. Pat. Nos. 5,834,177, and 5,945,271, which are incorporated herein be reference in their entirety. For example, eggs, larvae, nymphs or adults with predacious stages are provided with the growth medium in an amount effective to support growth, and incubated under conditions and for a period of time for the eggs or larvae to mature into pupae or adults, for nymphs to mature into adults and for the adults to advance through reproductive stages. Preferably, the growth medium is presented in a suitable container such as a petri dish or multicell container. Organdy may be used to enclose cells to permit air flow and prevent escapes. Because of the mobility of first and second instar larvae of zoophagous arthropods and their predisposition for cannibalism, multi-cell or other compartmentalized containers are preferred to segregate developing larvae. The cells and/or diet may be covered with a membrane. The membrane covers may be formed from a variety of polymeric materials, including but not limited to paraffin, polyethylene, polypropylene, and Parafilm(copyright). Use of Parafilm(copyright) as a cover is particularly advantageous as this membrane may serve as a phagostimulant, and it keeps the diet from drying out and from being accessed by microbes. I have found that presentation of the medium in stretched Parafilm(copyright) membrane is advantageous for the smaller predators so the mouthparts of the insect can readily penetrate the Parafilm(copyright).
Diet of the Second Embodiment
The artificial diet or growth medium of the second embodiment of the invention comprises a plant-based diet which includes cooked egg yolk or cooked whole egg. Optional ingredients may be included as described in detail below and as illustrated in Example 2 (see Components B and C). This diet is particularly suitable for providing nutrients in amounts and proportions effective to support growth of phytophagous arthropods, including facultatively zoophagous phytophages.
Plant materials for insect rearing are described in Handbook of Insect Rearing, P. Singh and R. F. Moore, Elsevier Press, Amsterdam, Volumes I and II, 1985, 488 and 514 pages, which are herein incorporated by reference. Without being limited thereto, exemplary sources of plant materials for the diet include beans, grains, seeds or nuts, ground or blended. This includes meal from wheat, soy, corn, rice, barley, oats, buckwheat, various beans and peas (e.g., legumes such as navy beans, pinto beans, mung beans), nut flours (peanuts, cashews, macadamias), plant seeds (e.g. sunflower seeds).
A critical feature is that the artificial diet or growth medium include added cooked egg yolk or cooked whole egg. Conveniently, this is carried out by adding fresh egg yolk or fresh whole egg to the plant-based diet and autoclaving or flash sterilizing the mixture. Other preparation procedures are within the skill of the art.
This plant-based, cooked egg diet also includes liquid. As with the diet of the first embodiment, the liquid can comprise water, or other sources of water such as milk, medium from tissue culture or aqueous plant extracts, and may include additional water soluble nutrients or additives such as vitamins, minerals, antimicrobial agents, or preservatives.
Other adjuvants or supplements may also be incorporated into the medium to enhance the growth of the target arthropod or prevent the growth of microbial contaminants. For example, the growth medium may also include other nutrients, for example, purified amino acids, nucleic acids, and/or lipids (with or without emulsifying agents) such as plant oils, e.g., corn oil, safflower oil, soybean oil or animal fats such as lard. These nutrients may also have important metabolic and behavior-inducing characteristics, such as phagostimulatory nutrients, that is, an ingredient that stimulates the target arthropod to stimulate the complete feeding response.
A multivitamin mixture, e.g., Vanderzant vitamin mixture, other sources of vitamins, or sources of B-vitamins, may be added to supplement vitamins in the growth medium.
Preservatives and/or antimicrobial agents as discussed in detail above may also be included in the diet as desired.
Components B and C of Example 2, below, illustrate an exemplary formulation of the second embodiment of the invention. Component B is prepared by mixing together sources of plant materials, liquid, and egg yolk or whole egg, and autoclaving and cooling the mixture. Component C is prepared by mixing together liquid, a vitamin mix, and optionally a preservative and/or antimicrobial agent as desired. Next, Component C is added to cooled Component B, and the mixture is blended. The combination of Components B plus C comprises the diet of the second embodiment.
The diet is a stiff (non-runny) slurry composed of a range of particles from about 1 to 1000 xcexcm. The diet comprises approximately 55-85% liquid and 15-45% plant diet plus cooked egg. Preferably, the amount of the protein in the growth medium, that is, the combined amount of all protein in the medium should be approximately 3-15% of growth medium (total wet weight), the amount of the lipid should be about 1-10% of the growth medium (total wet weight), the amount of carbohydrate should be about 5-30% of the growth medium (total wet weight), and the amount of liquid in the growth medium (including added liquid and liquid contained in the other ingredients) should be in the range of about 55-85% of the growth medium (total wet weight). The fat and protein sources may be provided in combination such as by lipoproteins. The mixture may be supplemented with pure vitamins (about 0.01% to 0.1% of the total diet) or with 1-5% vitamins (which include sugar filler).
The pH of the medium can range from about 4.0 to 8.0. A preferred pH range is about 4.8 to 6.8.
Water activity of the growth medium of the second embodiment is about 0.800 to 0.995, preferably about 0.90 to 0.99. Viscosity can be anything from a loose slurry (such as pourable Cream of Wheat(copyright) or oatmeal) to a stiff slurry such as oatmeal that cannot be poured and does not seek its own level in a container). A preferred mixture is one that is a stiff slurry that can, with pressure, fill a container.
The medium can be readily packaged, for example in a membrane such as Parafilm(copyright), as described in detail above. The packaged medium can be sterilized or pasteurized. The growth medium can be readily freeze-dried and can be used in the freeze-dried form or used after reconstitution with liquid. Techniques for rearing phytophagous arthropods including facultatively zoophagous arthropods are as described above.
Diet of the Third Embodiment
The artificial diet or growth medium of the third embodiment of the invention comprises the growth medium of the first embodiment in combination with a plant-based diet which includes cooked egg yolk or cooked whole egg (diet of the second embodiment of the invention). Optional ingredients may be included as described herein and as illustrated in the examples. This diet is particularly suitable for providing nutrients in amounts and proportions effective to support growth of phytophagous arthropods, including facultatively zoophagous phytophages.
In brief, preparation of the diet is carried out by mixing together (a) a plant-based diet of phytophagous insects which includes cooked egg yolk or cooked whole egg (95-80% by weight) with (b) 5-20% (by weight) of the growth medium of the first embodiment of the invention.
Plant materials for insect rearing are as described above. A critical feature is that the plant-based portion of the diet include added cooked egg yolk or cooked whole egg. Conveniently, this is carried out by adding fresh egg yolk or fresh whole egg to the plant-based portion and autoclaving or flash sterilizing the mixture.
Other adjuvants or supplements may also be incorporated into the medium to enhance the growth of the target arthropod or prevent the growth of microbial contaminants. For example, the growth medium may also include other nutrients, for example, purified amino acids, nucleic acids, and/or lipids (with or without emulsifying agents) such as plant oils, e.g., corn oil, safflower oil, soybean oil or animal fats such as lard. These nutrients may also have important metabolic and behavior-inducing characteristics, such as phagostimulatory nutrients, that is, an ingredient that stimulates the target arthropod to stimulate the complete feeding response.
A multivitamin mixture, e.g., Vanderzant vitamin mixture, other sources of vitamins, or sources of B-vitamins, may be added to supplement vitamins in the growth medium.
Preservatives and/or antimicrobial agents as discussed in detail above may also be included in the diet as desired.
An exemplary formulation is described in detail, below, in Example 2. In brief, a preferred formulation and preparation procedure are as follows:
Component A is prepared which comprises the diet of the first embodiment. Component B is prepared by mixing together sources of plant materials, liquid, and egg yolk or whole egg, and autoclaving and cooling the mixture. Component C is prepared by mixing together liquid, a vitamin mix, and optionally a preservative and/or antimicrobial agent as desired. Next, Component C and Component A are added to Component B and the mixture is blended. The combination of Components A plus B plus C comprise the diet of the third embodiment.
The diet is a stiff (non-runny) slurry composed of a range of particles from about 1 to 1000 xcexcm. Preferably, the amount of the protein in the growth medium, that is, the combined amount of all protein in the medium should be approximately 3-15% of growth medium (total wet weight), the amount of the lipid should be about 1-10% of the growth medium (total wet weight), the amount of carbohydrate should be about 5-30% of the growth medium (total wet weight), and the amount of liquid in the growth medium (including added liquid and liquid contained in the other ingredients) should be in the range of about 55-85% of the growth medium (total wet weight). The fat and protein sources may be provided in combination such as by lipoproteins. As with the second embodiment, the mixture may be supplemented with pure vitamins (ca. 0.01% to 0.1% of the total diet) or with 1-5% vitamins (which include sugar filler).
The pH of the medium can range from about 4.0 to 8.0. A preferred pH range is about 4.8 to 6.8. A pH of 5.8 produced superior results for rearing Lygus as shown in
Example 2, below.
Water activity of the growth medium of the third embodiment is about 0.800 to 0.995, preferably about 0.90 to 0.99. Viscosity can be anything from a loose slurry (such as pourable Cream of Wheat(copyright) or oatmeal) to a stiff slurry such as oatmeal that cannot be poured and does not seek its own level in a container). A preferred mixture is one that is a stiff slurry that can, with pressure, fill a container.
The medium can be readily packaged, for example in a membrane such as Parafilm(copyright), as described in detail above. The packaged medium can be sterilized or pasteurized, as desired. The growth medium can be readily freeze-dried and can be used in the freeze-dried form or used after reconstitution with liquid. Techniques for rearing phytophagous arthropods including facultatively zoophagous arthropods are as described above.
Example 2, below, describes the rearing of Lygus on the growth medium of the third embodiment. The new diet surpassed the existing Lygus standard diet (Debolt diet) in terms of productivity, biomass accumulation, and every other biological and economic parameter.
Tests of the new diet of the third embodiment with L. hesperus against a control diet consisting of the phytophage diet of U.S. Pat. No. 5,945,271 showed that Lygus hesperus reared on the growth medium of the third embodiment developed at rates greater than those reared on the control diet; they produced more eggs per female, had longer survival after reaching the adult stage, and the dry weights of the females and males were significantly greater than those of the control diet.
Further, by use of the growth medium of the third embodiment, for the first time in the history of rearing insects on artificial diets, L. lineolaris were reared for multiple generations and with continual increase of biomass. Efforts have been made to rear this insect for the past 40 years with no success until this diet.
Additionally, preliminary tests have shown that lepidopteran larvae (Heliothis virescens, Helicoverpa zea, and Spodoptera exigua) readily accepted the diet. Fire ants readily accepted the freeze-dried form of the diet.
As a supplement, the diet improved production of the predator Geocoris punctipes.